Homologs of aprotinin produced from a recombinant host, process, expression vector and recombinant host therefor and pharmaceutical use thereof

ABSTRACT

Microbially produced aprotinin and aprotinin homologs used for treating patients suffering from an excess release of pancreatic elastase, serum elastase or leukocyte elastase.

This is a division of application Ser. No. 07/029,501, filed 3/23/87,now pending.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to aprotinin homologs and their production viarecombinant DNA technology.

The chemically synthesized DNA molecules as disclosed herein arecharacterized by the DNA sequence coding for new polypeptides orpolypeptides substantially agreeing in the amino acid sequence andcomposition of aprotinin or aprotinin homologs and having the biologicalactivity of aprotinin or of aprotinin homologs.

2. Background Information

Aprotinin is a well known peptide comprising 58 amino acids and havingthe ability to inhibit trypsin, chymotrypsin, plasmin and kallikrein.Aprotinin is a basic proteinase inhibitor derived from bovine organs andhas become a valuable drug, named Trasylol®, for the treatment ofvarious diseases like, e.g., hyperfibrinolytic hemmorrhage andtraumatic-hemorrhagic shock (see H. Fritz and G. Wunderer, (1983), DrugRes., 33, 479-494).

Recently it has been shown, that homologs of aprotinin with otheraminoacids in position 15, instead of lysine, are valuable proteinaseinhibitors having modified effects and efficacies in comparison toaprotinin (DE-OS No. 33 39 693; H. R. Wenzel et al, 1985, in Chemistryof Peptides and Proteins, Vol. 3). These aprotinin homologs have stronginhibitory effects on the elastases from pancreas and leukocytes, and oncathepsin G.

Such homologs of aprotinin can be used therapeutically in diseases inconnection with excessive release of pancreatic elastase (pancreatitis),serum elastase (artherosclerosis), leukocyte elastase in acute andchronic inflammations with damage to connective tissue, in damage tovessel walls, in necrotic diseases and degeneration of lung tissue.Equally important is the part played by lysosomal enzymes, in particularleukocyte elastase, in inflammatory reactions due to immunologicalprocesses, for example, rheumatoid arthritis.

Although aprotinin and aprotinin homologs can be obtained from bovineorgans and by semisynthetic conversion of the bovine trypsin inhibitor(Tschesche, M., Wenzel, M., Schmuck, R., Schnabel, E.,Offenlegungsschrift DE No. 33 39 693), the yields are relatively small.

It was perceived that the application of recombinant DNA and associatedtechnologies would be the effective way of providing the necessary largequantities of high quality aprotinin homologs. The goal was to produceaprotinin homologs biologically active, as products of recombinant DNAtechnology from a host organism.

Methods for the expression of heterologous DNA in a microorganism arenow known.

DNA coding for polypeptides of known amino acid sequences may beprepared by using the genomic DNA sequence, the cDNA sequence which iscomplementary to the mRNA or by choosing codons according to the geneticcode and preparation of a synthetic gene.

A partial DNA sequence of a bovine genomic clone from bovine pancreatictrypsin inhibitor gene was cloned by S. Anderson and I. B. Kingston,(1983), Proc. Natl. Acad. Sci. USA, 80, 6838-6842 to characterize agenomic clone for BPTI.

A larger segment of the bovine genome coding for BPTI and bovine spleeninhibitor II were recently sequenced and published by Kingston, I. B.and Anderson, S., (1986), Biochem. J., 233, 443-450.

SUMMARY OF THE INVENTION

It is object of the present invention to provide aprotinin homologs,nucleic acids encoding them, vectors incorporating the nucleic acids andcells transformed therewith and methods of obtaining aprotininhomologues.

For the present purpose it was most advantagous to choose codons forpreparing synthetic genes with a proper design and which promisewidespread application.

This is especially the case by constructing a synthetic master genecomprising DNA blocks or cassettes terminated by unique recognitionsites of restriction enzymes. Such a gene design allows easymodification or mutation of all DNA sequences within such DNA blocks.

Homologs of aprotinin were prepared by recombinant DNA technology. Suchhomologs of aprotinin, for example, Val-15-, Ile-15-, Leu-15-, Phe-15-and Ala-15-, Arg-15, Gly-15, Ser-15, Trp-15, Tyr-15, aprotinin alone orin combination with a substitution at position 52 by Glu, Leu, Val, Argor Thr have been found to be equivalent to the known aprotinin and itshomologs, which have Met at position 52 and are disclosed together withtheir production. The substitution of Met-52 allows production ofaprotinin and aprotinin homologs in which a genetically engineered fusedpolypeptide is cleaved by cyanogen bromide at Met in the fusedpolypeptides.

The synthetic DNA coding for such homologs, recombinant plasmidscomprising structural genes for expressing the homologs and E. colitransformed by the recombinant plasmids are also disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram indicating a design of a syntheticaprotinin gene.

FIG. 2a depicts DNA sequences for sixteen DNA fragments used forproducing synthetic aprotinin.

FIG. 2b depicts DNA sequences for three beta-blocks.

FIG. 3 depicts a DNA sequence of a master gene used for producingsynthetic aprotinin.

FIG. 4 schematically depicts the construction of a plasmid according tothe present invention.

FIG. 5 schematically depicts the construction of a plasmid according tothe present invention.

FIG. 6 schematically depicts the construction of a plasmid according tothe present invention.

FIG. 7 shows a photograph of a 7.5% SDS-Polyacrylamide-Gel afterelectrophoresis and staining.

FIG. 8 shows a photograph of isolation products after gelelectrophoresis.

FIG. 9 is a chromatogram of a renaturated aprotinin.

FIG. 10 is a photograph of a western blot of fractionated CNBr peptidesof a Lys 15 Glu 52-aprotinin-beta-galactosidase fusion peptide.

FIG. 11 are two graphs (dose curres) depicting a comparison of aprotininand Glu-52-aprotinin by trypsin inhibitory activity.

FIG. 12 is a photograph depicting the results of a 8% SDS polyacrylamidegel electrophoresis.

FIG. 13 is a chromatogram of renaturated of val-15 Glu-52 Aprotinin.

DETAILED DESCRIPTION OF THE INVENTION

In the following, the strategy for construction and selection of DNAfragments coding for aprotinin and aprotinin homologs is shown.

The known protein sequence and the genetic code of aprotinin andaprotinin homologs were used to determine a DNA sequence coding for suchpolypeptides.

The degeneracy of the genetic code permits substantial freedom in thechoice of codons for any given amino acid sequence.

All possible base substitutions among the codons designating the aminoacid sequence of this protein were determined. According to this, allpotential restriction sites located within the possible DNA sequenceswere determined.

The codon choice for master genes were guided by the followingconsiderations:

First, codons and fragments were selected, and fragment assembly wasdesigned, so as to avoid undue complementarity of the fragments.

Secondly, regions rich in A-T base pairing are avoided to overcomeproblems with premature termination of transcription.

Thirdly, restriction sites were chosen necessary for facilitatingverification of transformants or base substitutions by replacement ofappropriate fragments with other fragments so that one can produceeasily modifications of aprotinin, examine the relationship between thestructures and their activities.

Fourthly, a majority of the codons chosen are those preferred in theexpression of microbial genomes (see H. Grosjean and W. Fiers, Gene, 18(1982), 192-209; M. Gouy and C. Gautier, Nucleic Acids Research, 10,(1982), 7055-7074).

The principal design for synthetic aprotinin genes and their homologs isshown in FIG. 1.

The design of the synthetic master gene, consisting of four blocks(named α, β, γ, δ) surrounded by recognition sites for restrictionendonucleases, further allows easy modifications and alterations of DNAsequences (codon usage, mutations, protein engineering, (amplificationof the genes) for unfused and fused expressions.

The synthetic genes were constructed as follows:

The synthetic genes for aprotinin and aprotinin homologs wereconstructed via a master gene by the assembly of 15 purifiedoligonucleotides which have overlapping terminal regions (see FIG. 1).This construction was done in two steps. First, part A and part B of thegene were produced by hybridization, ligation and purification of DNAfragments 1, 2, 3, 4 and 6 for part A and DNA fragments 5, 7, 8, 9, 10,11, 12, 13, 14 and 16 for part B (FIG. 2). Second, part A and B of thegene were ligated and purified. For this construction we used materialsand methods which are described hereafter. The DNA sequence of themaster gene is shown in FIG. 3. It includes the initiation codon ATG,two termination codons, TAG and TAA, the 5' terminal restriction sitefor Eco RI and the 3' terminal restriction sites for Hind III and BamHI, and also the internal restriction sites for Apa I, Stu I, Sac II(Sst II). These sites especially the internal ones, facilitate thecloning of the coding sequence, the modification of the master gene byexchanging DNA fragments which codes for other amino acids or which haveanother codon usage. An amplification of the gene can be done easily byadding appropriate linker sequences. The total spectra of proteinengineering is possible with such a construction.

To construct genes for aprotinin homologues only a restriction fragmentwith the appropriate DNA sequence has to be exchanged. Sequences forsuch fragments which will code for amino acid alterations includingpositions 15 and 52 were given in FIG. 2b.

The recombinant plasmids were constructed as follows:

The plasmid chosen for experimental aprotinin cloning was pUC 8 (J.Vieira and J. Messing, (1982), Gene, 19, 259). This cloning vectorconsists of a pBR 322 derived ampicillinase gene and the origin of DNAreplication ligated to a portion of the lac Z gene which contains anarray of unique restriction enzyme recognition sites. When this vectoris introduced into lac E. coli, the transformants give rise to bluecolonies on appropriate indicator plates. Cloning DNA fragments into anyof the multiple restriction sites, for example between Eco RI and BamHI, inactivates the lac gene giving rise to white colonies. Plasmid pUC8 is commercially available from P-L Biochemicals (see below).

The expression plasmids were constructed as follows:

For expression of aprotinin and aprotinin homologs as a fusion protein aplasmid has been constructed in which the appropriate gene was fusedwith the carboxy terminus of the β galactosidase gene as it was shown insimilar experiments by U. Ruther and B. Muller-Hill, (1983), EMBOJournal, 2, p. 1791-1794. The parental plasmid pUR 278 has the singlecloning sites, Bam HI, Sal I, Pst I, Xba I and Hind III at the 3' end ofthe lac Z gene (see also German patent application No. P 3 309 501.9).Insertion of a coding DNA sequence in the proper cloning sites and inthe correct reading frame leads to a fusion protein of active βgalactosidase combined with the peptide encoded by the DNA.

The restriction sites Bam HI and Hind III of expression vector pUR 278were chosen for cloning the synthetic genes for aprotinin and aprotininhomologues in an expression vector. Therefore, it was necessary tomodify the aprotinin gene by adding a Bam HI site at the 5' Eco RI endof the gene and using the Hind III site at the 3' end (see also FIG. 6).

The following standard material and methods for recombinant DNA workwere used:

The herein described synthetic genes, recombinant plasmids andexpression vectors with such genes can be prepared and characterized bythe following material and methods.

Material Enzymes

Polynucleotid-Kinase (PNK), 5,5 units/μl No. 633 542;Boehringer-Mannheim GmbH Biochemica, P.O. Box 310120, D-6800 Manheim 31,FRG

DNA Polymerase; Klenow, Boehringer-Mannheim No. 104 523 T4 DNA ligase,0.9 units/μl; Boehringer-Mannheim No. 481 220

Restriction enzymes were purchased from Bethesda Research LaboratorieGmbH, Offenbacher Str. 113, D-6078 Neu-Isenburg 1, FRG;Boehringer-Mannheim, Biolabs, 32 Tozer Road, Beverly, Mass. 01915-9990U.S.A.

Calf intestinal alkaline phosphatase (CIP); Boehringer-Mannheim

Lysozyme; RNase A; Boehringer-Mannheim

Reagents

Gamma 32 P ATP; Amersham No. PB 10168

Alpha 32 P-dTTP; Amersham 167

ATP; Sigma No. A-6144; Sigma Chemie GmbH, Grunwalden Weg 30, D-8024Deisenhofen, FRG

Bis Acrylamide; Serva 29195; Feinbiochemica GmbH & Co., D-6900Heidelberg 1, Postfach 105260

Acrylamide; Serva and Bio-Rad 161-0103; Bio-Rad Laboratories GmbH,Dachauer Str. 364+511 P.O. Box 50-0167, D-8000 Munchen 50, FRG

TEMED; Serva 35925

Ammonium Persulfate; Serva 13375

Urea; BRL ultra pure 5505 UA

DE 52 (preswollen diethylaminoethyl cellulose); Whatman, Cat 4075-050 W.& R. Balston Ltd, Springfield Mill, Mardstone, Kent, GB

DTE; Serva 20697

Isopropyl-β-D-thiogalactoside (IPTG); Sigma I 5502

5-brom-4-chlor-indolyl-β-D-galactoside (X gal); Boehringer-Mannheim651745

N,N'-dimethylformamid; Merck 2 203 034: E. Merck, Frankfurter Str. 250D-6100 Darmstadt 1, FRG

EGTA

Saccharose; BRL 5503 UA

Diaminopimelin acid; Sigma D 1377

M 13-Dideoxynucleotide Sequencing System; New England Biolabs, Beverly,Mass., U.S.A. #408, #409 chloroform

isoamylalcohol

Thymidine; Serva 18600

Glucose D (+); Merck 8337

Tris; Merck 8382

Kaliumhydroxid; Merck 5033

Calciumchlorid; Merck 2382

Rubidiumchlorid; Sigma R 2252

Manganchlorid; Sigma M 3634

DMSO; Sigma D 5879

EDTA;

Potassium acetate;

SDS;

DNA

Plasmid pUC 8; 27-4916-xx Bam HI linker; Pharmacia P-L Biochemicals,Munzinger Str. 9, Postfach 5480, D-7800 Freiburg 1, FRG

Media and Antibiotics

Bacto-tryptone; Difco 0123-01

Bacto-yeast-extract; Difco 0127-01

Bacto-Agar; 0140-01

LB-Medium: (for 1 ltr) 10 g Bacto-Trypton, 5 g Bacto-yeast-extract, 10 gNaCl, adjust pH 7.5 with NaOH)

kappa 1776-Medium: (for 1 liter) 25 g Bacto-Trypton, 7.5 gBacto-yeast-extract, 1M Tris-HCl (pH 7.5) 20 ml, ad 950 ml, autoclaveand cool down, add sterile: 5 ml 1M MgCl 2, 10 ml 1% diaminopimelineacid, 10 ml 0.4% thymidine, 25 ml 20% glucose

YT-Medium: (for 1 liter) 8 g Bacto-Trypton, 5 g Bacto-yeast-extract, 5 gNaCl

Agar plates were prepared by adding 15 g Bacto-Agar to 1 ltr of theappropriate medium.

Indicator plates: To 1 liter autoclaved YT medium with 1.5% agar thefollowing solutions were added: 2 ml 0.1M IPTG, 2 ml of 2% X-gal in N,N'dimethylformamide and 2 ml 100 mg/ml ampicillin.

Antibiotics

Chloramphenicol; Boehringer Mannheim 634 433

Ampicillin; Serva 13397

Tetracycline; Serva 35865

Buffers and Solutions

20 μM ATP: in water

10 mM ATP: in water

10X PNK-Mix: 0.5M Tris-HCl (pH 7.6); 0.1M MgCl 2; 50 mM DTE; 1 mM EDTA

10X Ligase-Mix: 0.5M Tris-HCl (pH 7.4); 0.1M MgCl 2; 0.1M DTE; 10 mM ATP

10X SP-50: 100 mM Tris-HCl (pH 7.5); 100 mM MgCl 2; 500 mM NaCl; 10 mMDTT

10X SP-100: 100 mM Tris-HCl (pH 7.5); 100 mM MgCl 2; 1M NaCl; 10 mM DTT

10X SP-0: 100 mM Tris-HCl (pH 7.5); 100 mM MgCl 2; 10 mM DTT

1M TBE: 1M Tris; 0.83M Boric acid; 10 mM EDTA; pH 8.3

3X Formamide Dye Mix: 70% formamide; 20% glycerol; 1 mM EDTA; 0.33 mg/mlbromphenol blue; 0.66 mg/ml xylenecyanol FE; 0.66 mg/ml orange G

20X E-buffer: 0.8M Tris; 0.4M sodium acetate; 40 mM EDTA; pH 8.3

10X CIP-buffer: 0.5M Tris-HCl (pH 9.0), 10 mM MgCl 2, 1 mM ZnCl 2, 10 mMSpermidine

Transformation buffer: Prepared as follows, 15 g saccharose, 1 ml 3.5MKOH, 1 ml 1M CaCl 2, 2 ml 5.0 m RbCl bring to 50 ml with aqua bidest,adjust pH 6.2 with 10% acetic acid, add 1 ml 4.5M MnCl 2, adjust pH 5.8with 10% acetic acid, fill to 100 ml with Aqua bidest and filtersterile.

TE buffer: 10 mM Tris-HCl ph 8.0, 0.1 mM EDTA

10X NT-buffer:

Lysozyme mix: 50 mM glucose, 1 mM EDTA, 10 mM Tris-HCL ph 8.0

Phenol/Sevag: mixture of 1 volume 80% phenol and 1 volume Sevag(Chloroform: iso amylalcohol 24:1)

Standard Methods

Standard Methods for recombinant DNA work were used as described inManiatis et al, (1982), Molecular Cloning, Cold Spring HarborLaboratory, Cold Spring Harbor, U.S.A. with some modifications asdescribed hereinafter.

Standard Ethanol Precipitation

DNA pellets were dissolved or solutions were adjusted to 0.3M sodiumacetate, two volume parts of ethanol were added, incubated at -70° C.for 15 minutes and centrifugated. Pellets were washed twice with 80%ethanol and dried under vacuum.

Standard Phenol Extraction

Solutions were mixed thoroughly with phenol/sevag, volume ratio 1:1,centrifuged, the phenol phase was reextracted with 1/10 volume of bufferor water, the aqueous phases were pooled.

Standard Isolation of DNA Fragments after Polyacrylamide GelElectrophoresis

Small DNA fragments (up to 250 nucleotides) were separated by gelelectrophoresis and bands were made visible by autoradiography or UVlight (pre-coated TLC-plates Silgur-25, Macherey-Nagel GmbH & Co. KG,Postfach 307, D-5160 Duren). Bands were sliced out, mashed with asiliconized glass rod, eluted with about 400 μl TE buffer pH 8.0 at 42°C. for 18 hours. The material was centrifuged and the pellet reeluted at42° C. for 4 hours and centrifuged. Both supernatants were combined andpurified by anion-exchange-chromatography on small DE-52 pasteur pipettecolumns with 1M TEABC buffer. After lyophilization DNA was dissolved andlyophilized in water twice.

Standard Ligation

For standard ligation (fragment smaller than vector) a molar ratio of1:5 were used for vector:fragment. Final DNA concentration was 25 μg/ml.DNA was solved in a small amount of TE buffer, 10X-Ligase mix, T4 DNAligase were added, adjusted to 1X Ligase mix concentration (50 mMTris-HCl pH 7.4, 10 mM MgCl 2, 10 mM DTE, 1 mM ATP; standard volume 30ul). Reaction was performed at 14° C. for 16 hours.

Standard 5' Labelling of DNA Fragments

Dephosphorylated DNA (final concentration about 0.2 μM) was solved in 1XKinase buffer I (50 mM Tris-HCl pH 7.6, 10 mM MgCl 2, 5 mM DTE, 0.1 mMEDTA). Together with unlabelled ATP, gamma 32 P ATP (3000 Ci/mmol) wasadded. Final concentration of ATP was always larger than 1 μM. Reactionwas carried out with an 500-1000 fold excess of polynucleotid kinasecalculated on the unit definition and the DNA concentration, at 37° C.for 30 minutes. Reaction was stopped for phenol extraction. DNA wasprecipitated with ethanol, washed and dried.

Standard Restriction Endonuclease Digestion

Restriction endonuclease digestions were carried out mainly according tothe manuals of the producers. Purified salt free DNA was dissolved inbuffer (SP-0, SP-50 or SP-100 respectively to the enzyme used) anddigested with an appropriate amount of enzyme. Finally material wasphenol extracted and ethanol precipitated.

Standard Isolation of DNA Fragments after Agarose Gel Electrophoresis

DNA fragments were separated by agarose gel electrophoresis (see T.Maniatis et al, (1982), Cold Spring Harbor Laboratory, MolecularCloning) stained with ethidium bromide and cut out under long wave UVlight. Slices were put into a dialysis bag, filled with 0.5X E-buffer(volume ratio, buffer:gel slice as 1.5:1) and must be well surrounded bybuffer. The sealed bag, air bubble free, was placed into anelectrophoresis chamber filled with 0.5X E-buffer. Electrophoresis wascarried out for 30 minutes at 200 V, than polarity of the current wasreversed for 30 seconds to release the DNA from the wall of the dialysisbag. The buffer surrounding the gel slice was carefully removed andpurified further on DEAE cellulose or DE 52 columns (see above).

Standard Dephosphorylation of DNA

DNA completely digested and purified was dissolved in water and adjustedto 1X CIP-buffer (standard total volume 48 μl). Reaction was started at37° C. by addition of 1 μl (20 units) calf intestine phosphatase (CIP)after 30 minutes again 1 μl CIP was added. Reaction was stopped after 1hour by adding 5 μl of 50 mM EGTA and incubation at 65° C. for 10minutes. For dephosphorylation of DNA with blunt ends or recessed 5'termini, repeated incubations were done for 15 minutes at 37° C. and for15 min at 56° C., respectively. The DNA was extracted with phenol/sevagand precipitated with ethanol.

Autoradiography

Films: AGFA-Gevaert, Curix RP 1, 100 AFW, Kodak XAR 5, 165 1512 x-raydeveloper; AGFA G153, Kodak LX24 x-ray fixer; AGFA G353; Kodak AL4.

Standard Transformation Procedure

Transformations were done, using the procedure of D. Hanahan ((1983), J.Mol. Biol., 166, 557-580).

1 ml of a 20 ml overnight culture of the host strain inoculated with asingle colony and grown in kappa 1776 medium (37° C., shaker with 200μpm), was used to inoculate 100 ml of prewarmed (37° C.) kappa 1776medium.

This culture was cultivated under the same conditions. Cell growth wasstopped at 0.2 OD 500 nm. After cooling to 4° C. and centrifugation,cell pellet was well resuspended in 20 ml ice cold transformation bufferand incubated at 0° C. for 5 minutes. The suspension was centrifugedagain (3000 rpm, 4° C., 15 minutes) and resuspended in 4 ml ice coldtransformation buffer. After adding 7 μl DMSO to 200 μl aliquots cellswere incubated further at ice water for 15 minutes to 60 minutes. Tosuch an aliquot of competent cells, DNA solved in 20 μl TE was added andthe mixture incubated in ice water 20 minutes and at 42° C. for 3minutes 1 ml of preheated (37° C.) kappa 1776 medium was inoculated bysuch an aliquot and a cultivation at 37° C. for 1 hour was carried out.For plating the transformants, cells were spun down (3000 rpm, 15minutes, 4° C.), resuspended in YT medium and plated on indicatorplates. According to the expected number of transformants a certainamount of the suspension was used for plating.

Standard Rapid Analytical Plasmid Isolation

This procedure is a modification of the method from Birnboim and Doly,1979 Nucl. Acids Res. 7, 1513, (see also T. Maniatis et al, 1982). Fromeach transformant which should be analysed a 2 ml overnight culture isprepared (wooden tooth pick, 37° C., 16 hours, rotating wheel). 1.5 mlof the overnight culture was centrifuged for 1 minute at 12,000 g(Eppendorf centrifuge). Pellet was redissolved in a freshly preparedsolution of 2 mg lysozyme per ml lysozyme mix and than incubated at 20°C. for 5 minutes. The sample was incubated for 5 minutes on ice afteraddition of freshly prepared ice cold 0.2M NaOH which contains 1% SDS.For precipitation of chromosomal DNA and proteins 150 μl ice coldpotassium acetate pH 4.8 was added. After incubation for 5 minutes at 0°C. and centrifuged for 10 minutes with 12,000 g the plasmid containingsupernatant was transferred to a fresh tube and extracted withchloroform/isoamylalcohol (24:1). 500 μl isopropanol were added to theaqueous phase. Mixture was incubated at -20° C. for 30 minutes. Aftercentrifugation (10 minutes, 12,000 g) sediment was washed with 80%ethanol and dried briefly in a vacuum. This material is sufficient for 5to 6 different restriction analysis by gel electrophoresis.

Standard Purification of Oligonucleotides with Polyacrylamide GelElectrophoresis

Oligonucleotides (about 20 OD 260 nm) were dissolved in bufferedformamide (0.1M TBE) and separated electrophoretically on 7M urea, 20%polyacrylamide gels (Maxam and Gilbert, Meth. Enzymol., 65, 500-560,(1980)). Gels were put on fluorescenced thin layer plates and DNA weremade visible with UV light. Isolation and purification were performed asoutlined in the standard protocol for isolation of DNA afterpolyacrylamide gel electrophoresis (see above). The quality of thisprocedure was routineously checked by analytical 5' phosphorylation withgamma 32 P ATP and polyacrylamide gel electrophoresis.

Preparation of the Glu-52-Aprotinin from a β-GalactosidaseLys-15-Glu-52-Aprotinin Fusion Protein

Many proteins synthesized in large quantities in bacteria accumulate inan insoluble form (D. C. Williams, R. M. Van Frank, J. B. Burnett, W. L.Muth, (1982), Science, 215, 687). These insoluble proteins are calledinclusion bodies. They may usually be solubilized only with denaturantsand therefore could easily be purified from other cell proteins.

E. coli strain RR1 delta M15 (ATCC 35102) was transformed with plasmidpRK 48.1.1., which encodes the Glu-52-aprotinin β-Galactosidase genedownstream from an E. coli promotor, operator and ribosome binding site.Maximal accumulation of the 15-Glu-52-aprotinin β-Galactosidase fusionprotein was 20% of total cell protein. The inclusions generallylocalized at the polar or sub-polar regions, with large percentage ofnormal-length cells having one inclusion near each pole.

An E. coli strain RR1 delta M15 overnight culture were centrifuged andthe pellet was then resuspended in a breaking buffer. After sonificationthe cell-lysate was centrifuged for recovering the inclusion bodies. Theinclusion bodies were washed with 2M guanidinium hydrochloride. Thepurification steps were checked by SDS-polyacrylamid electrophoresesaccording to Laemmli (U.K. Laemmli, (1970), Nature 277, 680-685), FIG.8.

For recovering the intact Lys-15-Glu-52-aprotinin the inclusion bodiesmust be solubilized, cleaved by cyanogen bromide and the unfoldedGlu-52-aprotinin has to be folded.

The inclusion bodies could be solubilized in 6M guanidiniumhydrochloride containing a sufficient amount of DTT. After separation ofnonsolubilized parts the fusion protein is precipitated by dialysingagainst water containing 10 mM mercaptoethanol. The wet fusion proteinwas dissolved in 70% formic acid and was cleaved by cyanogen bromideaccording to Gross et al (E. Gross, B. Witkop, (1961), J. Amer. Chem.Soc., 83, 1510-1511).

The Glu-52-aprotinin was separated from the cyanogen bromide fragmentsof the β-Galactosidase by ion exchange chromatography and wassimultaneously refolded by the procedure according to Creighton (T. E.Creighton, Proceedings of Genex-UCLA Symposium, (1985), Kingstones, inpress) (FIG. 9). The active inhibitor could be detected by Western blotanalysis (FIG. 10).

The active fractions were concentrated by evaporation and were thendialysed against 0.1M NH₄ HCO₃. After lyophilization the inhibitor waspurified by HPLC on a high pore RP-18 column using a gradient of 0.1%TFA in buffer A and 0.1% TFA 60% CH₃ CN in buffer B.

The active fractions were pooled and the inhibitor was characterized bymicrosequencing with a gas phase sequencer according to Hewick (R. M.Hewick, M. W. Hunkapiller, L. E. Hood, W. I. Dreyer, (1981), J. Biol.Chem., 256, 7990-7997). The first 20 residues from the N-terminus areidentical with the expected inhibitor (Table 2). The amino acid analysisdemonstrate that the inhibitor has the expected amino acid composition(Table 1).

A comparison of aprotinin and Glu-52-aprotinin by trypsin inhibitoryactivity shows identical dose-response curves (FIG. 11).

All these experiments show that it is possible to produceGlu-52-aprotinin as a fusion protein in E. coli and isolate it aftercleavage and separation under renaturing conditions.

Preparation of Val-15-Glu-52-Aprotinin and Other Derivatives ofAprotinin

Val-15-Glu-52-aprotinin can be prepared in a similar way fromβ-Galactosidase fusion protein as described for Glu-52-aprotinin (FIG.12, 13). The inhibitory activity was measured by an elastase inhibitoryassay.

The inhibitor was characterized by amino acid analysis and N-terminalsequencing (Tables 1 and 2).

All other derivatives of aprotinin could be prepared in a similar way asdescribed for Glu-52-aprotinin and Val-15-Glu-52-aprotinin.

                  TABLE 1                                                         ______________________________________                                        Amino acid analysis of aprotinin, Glu-52-                                     aprotinin and Val-15-Glu-52-aprotinin                                         Amino                                                                         Acid    Aprotinin    Glu-52  Val-15-Glu-52                                    ______________________________________                                        Asp     4,75 (5)     4,92 (5)                                                                              5,10 (5)                                         Thr     2,90 (3)     2,91 (3)                                                                              2,85 (3)                                         Ser     0,98 (1)     1,01 (1)                                                                              0,95 (1)                                         Glu     2,90 (3)     4,30 (4)                                                                              4,30 (4)                                         Gly     5,92 (6)     5,91 (6)                                                                              6,30 (6)                                         Ala     6,00 (6)     6,00 (6)                                                                              6,00 (6)                                         Val     1,04 (1)     1,02 (1)                                                                              2,06 (2)                                         Met     0,95 (1)     --      --                                               Ile     1,29 (2)     1,30 (2)                                                                              1,35 (2)                                         Leu     2,10 (2)     2,01 (2)                                                                              2,01 (2)                                         Tyr     3,92 (4)     3,70 (4)                                                                              3,81 (4)                                         Phe     3,86 (4)     4,08 (4)                                                                              4,05 (4)                                         Lys     3,99 (4)     3,80 (4)                                                                              3,10 (3)                                         Arg     5,82 (6)     5,75 (6)                                                                              6,00 (6)                                         ______________________________________                                    

The amino acids were measured after the post column derivatisation witho-phthalaldehyde. Cys and Pro were not determined.

                  TABLE 2                                                         ______________________________________                                        Amino acid sequencing of Glu-52 and Val-15-Glu-                               52-aprotinin (N-terminal)                                                     ______________________________________                                        1. Glu-52-aprotinin; about 1 nmol of the substance was                        sequenced over 20 cycles:                                                      ##STR1##                                                                      ##STR2##                                                                     2. Val-15-Glu-52-aprotinin; about 1 nmol of the substance                     was sequenced over 20 cycles:                                                  ##STR3##                                                                      ##STR4##                                                                     ______________________________________                                    

Comparison Aprotinin/Glu-52-Aprotinin

Glu-52-Aprotinin obtained after cleavage with BrCN was compared withauthentic aprotinin for its inhibitory activity against porcine trypsin.Both substrates Pyrglu-Gly-Arg-pNA and Benzoyl-Arg-pNa were used fortrypsin determination.

The stock solution for aprotinin was 1 μg/ml, and for Glu-52-aprotinin0.6 μg/ml. 0-100-200-300-400-500 μl of this stock solutions were used inthe test with Benzol-Arg-pNA. In the test with Pyrglu-Gly-Arg-pNA,0-3-6-9-12-15 μl were used. Results are summarized in Table 3.

                  TABLE 3                                                         ______________________________________                                               Aprotinin        Glu-52-Aprotinin                                             Amount inhi-         Amount inhi-                                             bitor       ΔE/10                                                                            bitor     ΔE/10                             Substrate                                                                            per assay   minutes  per assay minutes                                 ______________________________________                                        Benzoyl-                                                                             0       ng      0.91   0     ng    0.91                                Arg-pNA                                                                              100             0.76   60          0.77                                       200             0.57   120         0.68                                       300             0.39   180         0.58                                       400             0.08   240         0.44                                       500             0.00   300         0.30                                Pyr-Glu-                                                                             0       ng      0.85   0     ng    0.84                                Gly-Arg-                                                                             3               0.62   1.8         0.67                                pNA    6               0.39   3.6         0.56                                       9               0.29   5.4         0.47                                       12              0.18   7.2         0.32                                       15              0.13   9.0         0.28                                ______________________________________                                    

These results indicate, that aprotinin and Glu-52-aprotinin exhibitidentical dose-response curves in both trypsin inhibition assays. Thisdemonstrates not only, that the Glu-52-aprotinin contains practically100% active molecules, but also that the equilibrium constants of thetrypsin-inhibitor complexes are in the same order of magnitude.

Western Blotting

Western blotting was carried out as described by Towbin et al. (H.Towbin, T. Staehelin, I. Gordon, (1979), Proc. Natl. Acad. Sci. USA, 76,4350-4354). The nitrocellulose blot was probed with rabbit polyclonalanti-aprotinin antibodies as primary and biotinylated donkey anti rabbitantibodies as secondary antibodies. Detection of immuncomplexes wasperformed using a streptavidin-biotinylated horseradish peroxidasecomplex with 4-chloro-1-naphthol as substrate as described in thesupplier's manual (AMERSHAM BUCHLER GmbH, Gieselweg 1, D-3300Braunschweig 1).

ELISA

Solid phase enzyme linked immunosorbent assay (ELISA) was performed inthe competitive mode using microtiter antibodies plates as described byMuller-Esterl et al (W. Muller-Esterl, A. Oettl, E. Truscheit, H. Fritz,Fresenius Z., Anal. Chem., (1984), 317, 718-719).

Amino Acid Sequence Determination

About 0.5-2 nmol of the protein were solubized in 30 μl TFA. The samplewas applied to a glass fiber filter which was pretreated with 3 mg ofpolybrene. The sequence analysis was performed by the gas phase proteinsequencer from APPLIED BIOSYSTEMS, Inc., 850 Lincoln Centre Drive,Forster City, Calif. 94404, U.S.A.) according to Hewick et al (R. M.Hewick, M. W. Hunkapiller. L. E. Hood, W. Dreger, (1981), I. Biol.Chem., 256, 7990-7997). The stepwise liberated amino acidphenylthiohydantoin derivatives were analysed using a cyano-HPLC column(DU PONT, Wilmington, Del., U.S.A.) and a separation system described byBeyreuther et al (K. Beyreuther, B. Biesler, J. Bowens, R. Dildrop, K.Neufer, K. Stuber, S. Zais, R. Ehring, P. Zabel, (1983, Modern Methodsin Protein Chemistry p. 303-325, Walter de Gruyter & Co., Berlin). AWATERS HPLC system, including a M 510 pump, a WISP 710B auto-injector, aLC-spectrophotometer M 481 and a SHIMADZU integrator C-R3A was used.

Acid Hydrolysis and Aminoacid Analysis

About 1 nmol of the protein is given in a pyrex tube to which was added200 μl 6M HCl constant boiling HCl containing 0.05% 2-mercaptoethanol(I. T. Potts Jr., (1969), Anal. Biochem., 131, 1-15). Tubes were sealedunder vacuum and incubated at 110° C. for 22 h. Hydrolysates werequickly dried, redissolved in 150 μl 0.2M sodium citrate buffer pH 2.2and filtered. Amino acid analysis were carried out with a BIOTRONIK LC5000 amino acid analyzer equipped with a fluorescence detector and aSHIMADZU C-R2AX integrator. Amino acids were quantified after reactionwith o-phthaldialdehyde essentially as described by Benson et al (J. R.Benson, P. E. Hare 1975, Proc. Natl. Acad. Sci. USA, 72, 619-622).

Standard Leukocyte Elastase Assay

Materials

Human leukocyte elastase was obtained from Elastin Products Company,Inc., P.O. Box 147, Pacific, Miss. 63069 U.S.A.

Methoxysuccinyl-L-alanyl-L-alanyl-L-prolyl-L-valin-p-nitroanilide--K.Nakajima, J. C. Powers, M. J. Castillo, B. M. Ashe and M. Zimmerman, J.Biol. Chem., 254, 4027 (1979)--was obtained from Fa. Bachem,Feinchemikalien AG, Hauptstr. 144, CH-4416 Bubendorf/Schweiz.

Procedure

To 550 μl of a mixture of 0.2M Tris-buffer pH 8.0 containing 0.05%"TWEEN 80" and 0.05M in calcium chloride and the solution of theinhibitor 5 μl of a solution obtained on dissolution of 1 mg of theenzyme in 100 ml of 50% ethylenglykol were added. The mixture wasincubated 30 min. at room temperature. Then 100 μl of a mixture of 6.5μl of the solution of 59 mgMethoxy-succinyl-L-alanyl-L-alanyl-L-prolyl-L-valine-p-nitroanilide in 1ml of dimethylsulfoxide and test buffer was added with stirring. Theincrease in optical density at 405 was recorded; %inhibition wasdetermined by multiplying the coefficient of the increases in theoptical densities of the inhibitor containing sample and the enzymecontrol with 100.

Inhibition of Trypsin (Assays)

Inhibition of trypsin was determined by means of either the substratebenzoyl-DL-arginine-p-nitroanilide (Merck 1670) orpyroglutamyl-glycyl-arginine-p-nitroanilide, which was synthesized fromcommercially available pyroglutamyl-glycine (SENN 6886) andarginine-p-nitroanilide×HBr (SENN 9123) by means of the dicyclohexylcarbodiimide condensation method. This substrate is also sold under thedesignation S-2444 by KABI as an urokinase substrate.

The former has the advantage of giving a linear response to the amountof Aprotinin in the sample, but has a low sensitivity. The latter onehas a high sensitivity, but due to the dissociation of theaprotinin-trypsin-complex at those low concentrations, thedose-response-curve is non linear.

The buffer for the measurement of trypsin and trypsin inhibitors is 0.2MTris, pH 8.0 containing 0.01M CaCl₂ and 0.05% Tween 80®.

The determinations can be performed in any spectral photometer whichallows readings of optical densities (ODs) at 400 nm. For fullyautomated measurements, the photometers have to be equipped with a microprocessor or must be interfaced with a suitable personal computer.

Disposable 1 cm semi micro plastic cuvettes are used for all assays. ODsare read in time intervals of 1 minute over 8 cycles at ambienttemperature. The average increase of OD per minute is arbitrarily takenas the activity unit.

For inhibition assays, porcine trypsin (Merck 8350) solution (in 0.001NHCl/50% glycerol) is mixed with the inhibitor sample, adjusted to 500 μlwith buffer and incubated for 10 minutes at ambient temperature. Thereaction is initiated by the addition of substrate solution. Moredetailed information is given in the Table 4 hereinbelow. Inhibitoryactivities are taken from a calibration curve or automaticallycalculated by computer programs developed especially for this purpose.

                  TABLE 4                                                         ______________________________________                                               Assay with Pry-Glu-Gly-                                                                       Assay with Benzoyl-                                           Arg-pNA as the sub-                                                                           Arg-pNA as                                                    strate          the substrate                                          ______________________________________                                        Trypsin  15 μl (1 μg/ml)                                                                           20 μl (100 μl/ml)                            Substrate:                                                                             50 μl (0.02 M in                                                                             50 μl (0.012 M in                                        10% ethanol)      10% DMSO)                                          ______________________________________                                    

A large number of various microorganisms are known in the art as beingsuitable for transformation. That is, those unicellular organisms whichare capable for being grown in cultures or fermentation. Preferredorganisms for transformation include bacteria, yeasts and fungi.

The particular organism chosen for the work disclosed here, was E. coliRR1ΔM15, which has been deposited with the American Type CultureCollection, ATCC No. 35102. Other suitable E. coli strains may also beemployed.

The present invention includes pharmaceutical preparations which inaddition to non-toxic, inert pharmaceutically suitable excipientscontain one or more compounds according to the invention or whichconsist of one or more active compounds according to the invention, andprocesses for the production of these preparations.

The present invention also includes pharmaceutical preparations indosage units. This means that the preparations are in the form ofindividual parts, for example tablets, coated tablets, capsules,caplets, pills, suppositories and ampoules, of which the content ofactive compound corresponds to a fraction or a multiple of an individualdose. The dosage units can contain, for example, one, two, three or fourindividual doses or one half, one third or one quarter of an individualdose. An individual dose preferably contains the amount of activecompound which is given in one administration and which usuallycorresponds to a whole, a half or a third or a quarter of a daily dose.

By non-toxic, inert pharmaceutically suitable excipients there are to beunderstood solid, semi-solid or liquid diluents, fillers and formulationauxiliaries of all kinds.

Tablets, coated tablets, capsules, caplets, pills, granules,suppositories, solutions, suspensions and emulsions, pastes, ointments,gels, creams, lotions, powders and sprays may be mentioned as preferredpharmaceutical preparations.

Tablets, coated tablets, capsules, caplets, pills and granules cancontain the active compound or compounds alongside the customaryexcipients such as (a) fillers and extenders, for example, starches,lactose, sucrose, glucose, mannitol and silica, (b) binders, forexample, carboxymethylcellulose, alginates, gelatin andpolyvinylpyrrolidone, (c) humectants, for example glycerol, (d)disintegrants, for example, agar-agar, calcium carbonate and sodiumcarbonate, (e) solution retarders, for example, paraffin and (f)absorption accelerators, for example, quaternary ammonium compounds, (g)wetting agents, for example, cetyl alcohol and glycerol monostearate,(h) adsorbents, for example, kaolin and bentonite and (i) lubricants,for example, talc, calcium and magnesium stearate and solid polyethyleneglycols, or mixtures of the substances listed under (a) to (i).

The tablets, coated tablets, capsules, caplets, pills and granules canbe provided with the customary coatings and shells, optionallycontaining opacifying agents, and can also be of such composition thatthey release the active compound or compounds only, or preferentially,in a certain part of the intestinal tract, optionally in a delayedmanner, examples of embedding compositions which can be used includepolymeric substances and waxes.

The active compound or compounds, optionally together with one or moreof the abovementioned excipients, can also be in a microencapsulatedform.

Suppositories can contain, in addition to the active compound orcompounds, the customary water-soluble or water-insoluble excipients,for example, polyethylene glycols, fats, for example, cacao fat andhigher esters (for example, C₁₄ alcohol with C₁₆ fatty acid) or mixturesof these substances.

Ointments, pastes, creams and gels can contain the customary excipientsin addition to the active compound or compounds, for example, animal andvegetable fats, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silica, talcand zinc oxide, or mixtures of these substances.

Powders and sprays can contain the customary excipients in addition tothe active compound or compounds, for example, lactose, talc, silica,aluminium hydroxide, calcium silicate and polyamide powders, or mixturesof these substances. Sprays can additionally contain the customarypropellants, for example, chlorofluorohydrocarbons.

Solutions and emulsions can contain the customary excipients in additionto the active compound or compounds, such as solvents, solubilizingagents and emulsifiers, for example water, ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide,oils, in particular cotton seed oil, groundnut oil, corn germ oil, oliveoil, castor oil and sesame oil, glycerol, glycerolformal,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, or mixtures of these substances.

For parenteral administration, the solutions and emulsions can also bein a sterile form which is isotonic with blood.

Suspension can contain the customary excipients in addition to theactive compound or compounds, such as liquid diluents, for examplewater, ethyl alcohol or propylene glycol, suspending agents, forexample, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol andsorbitan esters, microcrystalline cellulose, aluminium metahydroxide,bentonite, agar-agar and tragacanth, or mixtures of these substances.

The formulation forms mentioned can also contain dyestuffs,preservatives and additives which improve the odour and flavour, forexample, peppermint oil and eucalyptus oil, and sweeteners, for example,saccharin.

The therapeutically active compounds should preferably be present in theabovementioned pharmaceutical preparations in a concentration of about0.1 to 99.5, preferably of about 0.5 to 95, percent by weight of thetotal mixture.

The abovementioned pharmaceutical preparations can also contain otherpharmaceutical active compounds in addition to the compounds accordingto the invention.

The abovementioned pharmaceutical preparations are manufactured in theusual manner according to known methods, for example, by mixing theactive compound or compounds with the excipient or excipients.

The active compounds or the pharmaceutical preparations can beadministered locally, orally, parenterally, intraperitoneally and/orrectally, preferably orally or parenterally, such as intravenously orintramuscularly.

In general, it has proved advantageous both in human medicine and inveterinary medicine to administer the active compound or compoundsaccording to the invention in total amounts of about 0.5 to about 500,preferably 5 to 100, mg/kg of body weight every 24 hours, optionally inthe form of several individual administrations, in order to achieve thedesired results. An individual administration contains the activecompound or compounds according to the invention preferably in amountsof about 1 to about 250, in particular 3 to 60, mg/kg of body weight.However, it can be necessary to deviate from the dosages mentioned and,in particular, to do so as a function of the nature and body weight ofthe subject to be treated, the nature and severity of the illness, thenature of the preparation and of the administration of the medicine, andthe time or interval over which the administration takes place.

Thus, it can suffice in some cases to manage with less than theabovementioned amount of active compound, whilst in other cases theabovementioned amount of active compound must be exceeded. Theparticular required optimum dosage and the type of administration of theactive compounds can easily be decided by anyone skilled in the art onthe basis of his expert knowledge.

E. coli transformed with expression plasmids pRK 49.2.1 and pRK 48.1.1were deposited with Deutsche Sammlung von Mikroorganismen, Grisebachstr.8, D-3400 Gottingen under the deposit numbers DSM 3678 and DSM 3679.

The Arg-15-homologues as for example the homologue of Example 7 are veryuseful in the treatment of acute inflammatory diseases as for examplerheumatoid arthritis, hereditary angioneurotic edema, pneumonitis,pancreatis and shock. The Arg-15-homologues are further useful asprotective agents in dialysis procedures involving artificial membranesand in extracorporal circulation.

EXAMPLES EXAMPLE 1 Synthesis and Purification of DNA Fragments Codingfor Glu-52- and Val 15-Glu-52-Aprotinin

The oligonucleotides which comprise the gene were prepared usingsolid-phase synthetic methods. The synthetic scheme for the oligomerswas as outlined and utilized proton activated, protected2'-deoxyribonucleotide phosphoramidites. All sequential steps wereperformed in an automated manner on an Applied Biosystems Model 380 DNASynthesizer using protected nucleotides, solvents, chemicals andreagents obtained from this manufacturer. The solid-phase support, alsofrom the same manufacturer, was controlled pore glass to which thestarting 3'-nucleotide was already attached. Certain modifications wereintroduced into the automated reaction cycle in accordance with theManufacturer's Operating Instructions and Users' Bulletins. Uponcompletion of the synthesis, the oligomers were deblocked and cleavedfrom the solid support within the DNA synthesizer according to themanufacturer's recommendations.

Removal of the blocking groups was completed by heating the aqueoussolution containing the oligomer with concentrated ammonium hydroxide at55° C. from 4 to 24 hours in a sealed vial. The resulting solution wasevaporated, the residue dissolved in 0.01M triethylammonium bicarbonatebuffer, pH 7.0 (TEAB buffer). This solution was chromatographed overSephadex-G 50® Gel Filtration Resin. This column was prepared in andeluted with the same TEAB buffer. Material eluting with the void volumewas pooled and the solution evaporated.

A portion of the residue (10 to 40% of the absorbance units at 260 nm),dissolved in loading buffer (composition: 0.1% Bromophenol Blue, 0.1%xylene cyanol, 10 mm disodium EDTA, in formamide) was further purifiedby electrophoresis on polyacrylamide gels. The gel size was 18×32 cmwith a thickness of 1.5 mm. The well size for each oligomer purified inthis manner was 2 to 5 cm in width and up to five oligomers werepurified using a single gel. The concentration of acrylamide in the gelvaried from 14 to 20%, depending on the chain length of the desiredproduct. For longer oligomers, a 14% acrylamide gel is preferred, whileshorter oligomers were purified on up to a 20% acrylamide gel. The gelsalso contained 7M urea and Tris-borate-EDTA buffer (0.1M Tris, 0.1MBorate, 2 mM EDTA, pH 8.3). The running buffer was the sameTris-borate-EDTA mixture. Electrophoresis was carried out at 20 to 60watts, constant power, for from 18 to 6 hours. Such standardizedtechniques are available in various User Information Bulletins availablefrom Applied Biosystems.

Following completion of the electrophoresis, the gel was enclosed inplastic wrap and the oligomers visualized by shadowing with ultravioletlight. This shadowing was accomplished by placing the wrapped gel on afluorescent thin layer chromatography plate and viewing the gel with ashort wave length ultraviolet light source. The desired product appearedas the slowest migrating, major blue DNA fragment by this shadowingtechnique. The desired band was exised from the gel. The DNA oligomerwas eluted from the gel slice onto powdered diethylaminoethyl (DEAE)cellulose using an EpiGene D-Gel® electrophoresis apparatus. Theoligomer was recovered from the cellulose by elution with 1M TEABbuffer. The buffer solution containing the oligomer was evaporated, theresidue was dissolved in 0.01M TEAB buffer, and then desalted by passageover a column of Sephadex-G 50® as described previously. The materialeluting in the void volume was pooled and lyophilized to give the finalproduct.

Using the procedures outlined above, about 0.5 to 5.0 A260 units of eachof the purified oligomers was obtained.

EXAMPLE 2 Construction of a Synthetic Aprotinin Master Genes for Glu-52-and Val-15-Glu-52-Aprotinin

The construction of these specific synthetic aprotinin genes involve theassembly of 15 purified oligonucleotides (see FIG. 14). The DNA sequenceshown in FIG. 3, includes the initiation codon ATG, two terminationcodons, TAG and TAA, the terminal restriction sites Eco RI, Hind III andBam HI and internal restriction sites. The choice of these sitesfacilitated the cloning of the coding sequence and its modification.

The construction used to generate this synthetic gene employed besidesthe fragments the use of Polynucleotid Kinase, T4 DNA ligase andrestriction enzymes as described in detail within material and methods.

Fifteen purified oligonucleotide fragments were dissolved in 50 mM TEABC(triethylammonium bicarbonate buffer, pH 7.5), final concentration 10pmol/μl. The phosphorylation of all fragments was done in 4 separateparts (Frag. 1,3; Frag. 2,4,6; Frag. 5,7,9,11,13; Frag. 8,10,12,14,16).For preparative purposes, 80 pmol of each fragment, respectively, weredissolved in a mixture of 1X PNK-Mix, 2 μM ATP, 0.5 uCi 32 P gamma ATPper 10 pmol fragment, 10 units PNK per pmol fragment, so that the totalvolumes were for Frag. 1,3; 300 μl, for Frag. 2,4,6; 400 μl, for Frag.5,7,9,11,12; and Frag. 8,10,12,14,16; 700 μl. Reaction for each part wascarried out at 37° C. for 30 minutes. All parts were phenolized, ethanolprecipitated, washed and dried.

For hybridization purposes, Frag. 1,3 and Frag. 2,4,6 (block A) weredissolved and mixed in 1X Ligase-Mix; total volume 120 μl, incubated for5 minutes at 70° C. and cooled down to room temperature within 5 hours.The other fragments (block B) were hybridized in 240 μl according to thesame procedure.

For ligation purpose, block A solution was supplemented with 12 μl 10 mMATP, 12 μl 100 mM DTE, 20 μl TADNA ligase and block B solution withtwice as much. Reaction was carried out at 14° C. for 7 hours. Afterthis 10 μl T4 DNA ligase was added for block A and 20 μl for block B andagain incubated at 14° C. for 45 minutes. The mixtures were phenolized,ethanol precipitated and dried.

The obtained block A was dissolved in 90 μl 1X SP-100 and 10 μl Eco RI(10 μ/μl), block B in 90 μl 1X SP-50 and 10 μl Bam HI and incubated at37° C. for 1 hour. The reactions were stopped by phenol extraction andethanol precipitation, 6% polyacrylamide gel electrophoresis was carriedout, and the DNA blocks were recovered according to the same procedureas described above.

Equal amounts of radioactive labelled block A and B were dissolved inwater, adjusted to 1X ligase mix and hybridized as described above forfinal ligation to a synthetic gene. Therefor, 3 μl 10 mM ATP, 3 μl 100mM DTE, 3 μl T4 DNA ligase were added to 22 μl of a hydridizationmixture and incubated at 14° C. for 7 hours. Again 1 μl T4 DNA ligasewas added and this reaction was carried out at 14° C. for 45 minutes.The ligation product was purified by phenol extraction and ethanolprecipitation. A standard restriction enzyme digestion (Bam HI 1.5 μl,Eco RI 1.5 μl double digestion) in SP-50 was performed. The material wasphenol extracted and before ethanol precipitation the aqueous solutionwas adjusted to 3 mM MgCl₂ 0.3M sodium acetate. Then, 6% polyacrylamidegel electrophoresis was carried out, and the gene was recoveredaccording to the same procedure as described above.

EXAMPLE 3 Construction of Recombinant Plasmids pRK 63.1.1 and pRK 54.1.1

The plasmid chosen for experimental aprotinin cloning was pUC 8 (J.Vieira and J. Messing, (1982), Gene, 19, 259). This cloning vectorconsists of a pBR 322 derived ampicillinase gene and the origin of DNAreplication ligated to a portion of the lac Z gene which contains anarray of unique restriction enzyme recognition sites. When this vectoris introduced into lac E. coli, the transformant give rise to bluecolonies on appropriate indicator plates. Cloning DNA fragments into anyof the multiple restriction sites, for example between Eco RI and BamHI, inactivates the lac gene giving rise to white colonies.

Vector Preparation

For ligating the synthetic aprotinin master gene into pUC 8, apreparative vector preparation was performed. Purified pUC 8 DNA (about30 pmol) were digested twice with Eco RI and Bam HI under standardrestriction endonuclease digestion conditions, to cut out a smallinternal Eco RI-Bam HI fragment. This preparation was dephosphorylatedwith calf intestine phosphatase as described above, separated by agarosegel electrophoresis and the large Eco RI-Bam HI fragment of the vectorwas purified (standard conditions). This procedure facilitatesenormously the further work with the vector, because self ligation ofvector molecules at the Eco RI and Bam HI termini are excluded and thebackground of transformants is reduced drastically.

Ligation

The construction of pRK 63 (see FIG. 4) was done by ligating the totalamount of purified synthetic aprotinin gene with 1 pmol vector (1.8units T4-DNA-ligase, 1X ligase mix, total volume 45 μl, incubation at14° C. for 7 hours, addition of 1 unit T4-DNA-ligase and reincubation at14° C. for 45 minutes).

Transformation

Using the transformation procedure from D. Hanahan, supra (for detailssee the standard transformation procedure) E. coli strain RRI delta M15(A. Kalnins et al, (1983), EMBO Journal 2, 593; ATCC 35102) was used asreceptor cell. 15 "white" transformants were received aftertransformation with 50% of the ligation material on indicator platescontaining 200 μg/ml ampicillin. All 15 transformants were screenedusing a modification of the rapid analytical plasmid isolation method ofBirnboim and Doly (1979) (see above). Therefore, pellets of the 15samples were redissolved in 30 μl 1X SP-100 containing 1 μg RNase A. Arestriction digestion with Eco RI and Bam HI was performed.

After gel electrophoresis four of the fifteen transformants were foundto contain plasmid DNA carrying an Eco RI-Bam HI fragment approximately200 base pairs long.

All transformants which carried this Eco RI-Bam HI fragment were grownin large scale and plasmids from each were isolated and analysedfurther. Two of them were sequenced according to the procedure of Maxamand Gilber (Proc. Natl. Acad. Sci. U.S.A. (1977) 74, 560-564) all showedthe correct sequence, demonstrating the excellence of chemical synthesisand construction.

Plasmid pRK 54.1.1 (Val-15-Glu-52 aprotinin) was constructed by a simpleexchange of the beta block of the synthetic gene, which is an Apa I-StuI fragment, with a beta block containing a codon for Val at position 15instead of Lys.

About 100 pmol of the synthetic ss DNA fragments BEA 4A and BEA 4B (seeFIG. 2B) were dissolved in 20 μl water, heated for 5 minutes at 95° C.and cooled down slowly to room temperature (5 hours). The hybridizedunphosphorylated fragment was ligated with 1.5 pmol purified DNA frompRK 63.1.1. missing the Apa I-Stu I fragment. Standard ligation was done30 μl ligation mix. Transformation of E. coli RRIΔM15 was done with 50%of the ligation mixture. From 1500 transformants 24 were tested by ananalytical plasmid isolation and restriction analysis. All were positiveand two of them were sequenced by the M 13 Dideoxynucleotide SequencingSystem from BioLabs, Beverly, Mass., U.S.A. The transformant pRK 54.1.1were used for further experiments.

EXAMPLE 4 Construction of Expression Plasmids pRK 48.1.1 and pRK 49.2.1

For expression of aprotinin as a fusion protein a plasmid wasconstructed in which the aprotinin gene was located at the carboxyterminus of the β-galactosidase gene as it was shown in similarexperiments by U. Ruther and B. Muller-Hill, (1983), EMBO Journal, 2,1791-1794 and in German Pat. appl. DE-OS No. 33 09 501.9.

For cloning the synthetic aprotinin gene in expression vector pUR 278cloning sites Bam HI and Hind III were chosen. Therefor, it wasnecessary to modify the aprotinin gene by adding a Bam HI site at the 5'Eco RI end of the gene and using the Hind III site at the 3' end (seealso FIG. 6).

50 pmol pRK 63.1.1 DNA were completely digested overnight at 37° C. withEco RI (15 pmol hit/μl) in 120 μl 1X SP-100. The protruding 5' Eco RIends of this DNA material were filled by an enzymatic reaction with DNApolymerase I (Klenow fragment), dATP and dTTP (Maniatis et al, (1982),supra) 600 pmol of dried alpha 32 P ATP (250 uCi) were solved and mixedwith 160 μl DNA (50 pmol), 10 μl 1 mM dATP (10 000 pmol), 20 μl NTbuffer. Than 10 μl DNA polymerase I (Klenow, (50μ) were added and afirst incubation at room temperature took place. After 30 minutes, 10 μl1 mM dTTP (10 000 pmol) were added together with 5 μl polymerase (25μ)and the second incubation at room temperature took place. The materialwas phenol/sevag extracted, molecular weight fractions radioactivelabelled, were pooled, ethanol precipitated, washed, solved in 50 μl TEand stored at 20° C.

20 μl of this material with flush ends were used for ligation with BamHI linker. Therefore, 400 pmol of 5' Bam HI linker labelled with gamma32 P ATP (standard 5' phosphorylation procedure) were ligated to 40 pmolDNA ends (standard ligation conditions, 4.5μ T4 DNA ligase, total volume60 μl). To control the quality of linker ligation an analytical gelelectrophoresis were performed. Complete ligation was achieved afteradding 100 pmol Bam HI linker, 1.8 units T4 DNA ligase, incubation at20° C. for 1 hour and adding 1 unit T4 DNA ligase and incubation at 14°C. for 18 hours. The reaction mixture was phenol/sevag extracted,ethanol precipitated, washed, dried and solved in 40 μl TE.

For preparation of the synthetic aprotinin gene with Bam HI and Hind IIItermini, the linkered linear plasmid (10 pmol) was cut first with HindIII (10.5 pmol hit/μl, 5 hours, 37° C.), and then with Bam HI (40 pmolhit/μl, 20 hours, 37° C. standard conditions). The fragment was isolatedafter separation on 6% polyacrylamide gel electrophoresis and carefullypurified (see standard procedure).

Vector Preparation

The parental vector pUR 278 (about 5 pmol) was cut first with Hind III(standard conditions) purified by phenol/sevag extraction, ethanolprecipitation, redissolved and then digested with Bam HI (standardconditions). This material was loaded on a 1% agarose gel,electrophorized, isolated and purified according to the standardconditions, to get rid of the 18 base pair long Bam HI-Hind III fragmentwhich would compete in ligation with the synthetic aprotinin gene.

Ligation and Tranformation

For ligation, a 0.3 pmol vector, 1.5 pmol fragment (approximately), 2units T4 DNA ligase were used (standard conditions: total volume 30 μl,incubation: 4 hours at 14° C.).

Transformation was performed with E. coli strain RR1 delta M 15 as hostusing one third of the ligation mixture (standard conditions). A totalof 173 "blue" colonies were received on indicator plates containing 200μg ampicillin/ml. From this 12 transformants were analysed further byrapid analytical plasmid isolation (standard conditions). Of 173transformants 30 should be background transformants, calculated on thepercentage of transformants received by religation of vector. Thisresult was confirmed by restriction analysis of plasmids of the 12transformants. 8 of them were positive showing a Bam HI-Hind IIIrestriction fragment of about 200 base pairs. Positive recombinantplasmids were also linearized by Sst II and unique restriction sitewithin the aprotinin gene. Base sequence analysis according to Maxam andGilbert, 1980, revealed that the plasmid pRK 48.1.1 has inserted thedesired aprotinin DNA fragment (see FIG. 6). Plasmid pRK 48.1.1 was usedfor further analysis and expression work.

The construction of plasmid pRK 49.2.1 was done by exact the sameprocedure using the Val-15-Glu-52 gene from pRK 54.1.1. The positiverecombinant plasmid 49.2.1 showed the correct DNA sequence and thisconstruction was used for further analysis and expression work.

Detection of Expression of β-Galactosidase-Glu-52-Aprotinin andβ-Galactosidase-Val-15-Glu-52-Aprotinin

To attempt expression of each of these constructions, E. coli strainswith pRK 48.1.1 (deposited at DSM, DSM No. 3679) and pRK 49.2.1(deposited at DSM, DSM No. 3678) were inoculated into 2 ml LB-ampicillinmedium supplemented with 4 μl of 0.1M IPTG. A clone containing pUR 278without aprotinin gene insert was also inoculated into culture medium toprovide for the a negative control for the assays. After 12-16 hoursgrowth at 37° C. with agitation samples of 1 ml were used directly forinoculation of 100 ml LB-ampicillin medium. After growing for 12-16hours at 37° C. with agitation, the cells were harvested bycentrifugation at 5000 rpm for 10 minutes in a Beckmann JA 10 rotor.

Direct detection of fusion proteins were performed with SDSPolyacrylamide gel electrophoresis according to Laemmli, U.K., (1970),Nature, 277, p. 680, see also B. D. Hames and D. Rickwood, (1981), GelElectrophoresis of Proteins, IRL Press Limited, Oxford, England).

Per lane about 1×10E9 cells were centrifuged and redissolved in a 1:5dilution of SDS sample buffer (0.3M Tris HCL pH 8.8, 50% glycerol, 5%SDS, 25% mercapto ethanol). After electrophoresis gels were stained withCoomassie blue. FIG. 7 shows a typical pattern of E. coli proteins withthe inducible β-galactosidase Glu-52 aprotinin fusion protein.

Solutions

Breaking buffer:

50 mM Tris

100 mM Sodium chloride;

10 mM Magnesium chloride;

10 mM Mercaptoethanol;

2M Guanidinium-HCl-solution:

2M Guanidinium-HCl;

50 mM Tris-HCl, pH 7.7

100 mM Sodium chloride;

10 mM Mercaptoethanol;

6M Guanidinium-HCl-solution:

6M Guanidinium-HCl;

50 mM Tris-HCl;

100 mM Sodiumchloride;

10 mM Mercaptoethanol.

EXAMPLE 5 Preparation of Glu-52-Aprotinin 1. Isolation and CyanogenBromide Cleavage of β-Gal-Fusion Protein Lys-15-Glu-52-Aprotinin

For preparation purposes, 6 liters of an E. coli overnight culturestrain RR1 delta M15 were centrifuged for 15 minutes at 8000 rpm. Thecell pellet, about 15 g in weight, was resuspended in 30 ml of breakingpuffer and sonified for 6 minutes (ice cooling). The cell lysate wascentrifuged for 20 minutes at 20000 rpm. The supernatant was discarded.The pellet, about 10 g was resuspended in 20 ml of 2M guanidiniumhydrochloride solution and was homogenized. After centrifugation for 20minutes at 2000 rpm, the supernatant was discarded. The pellet, about 8g, was dissolved under a N₂ atmosphere in 20 ml 6M guanidiniumhydrochloride solution containing 200 mg Dithiothocitol and reduced for1 hour at 50° C. The solution was centrifuged for 10 minutes at 10000rpm and dialysed for 24 hours against water containing 10 mMmercaptoethanol. The precipitated fusionprotein was collected bycentrifugation for 20 minutes at 20000 rpm. The wet fusion protein wasdissolved in 30 ml conc. formic acid and then diluted to 70% with water.The fusion protein was cleaved by adding 4 g of cyanogen bromide andincubation for 18 hours under nitrogen atmosphere in the darkness. Thereaction was stopped by diluting with 200 ml water. The water and thevolatile by-products were removed by freeze drying. The cyanogen bromidecleavage was checked by SDS gel electrophoresis according to Laemmli,supra. The yield was about 1.5 g of cyanogen bromide fragments. In aseries of experiments the yields varied from 0.8 to 2.5 g.

Solutions

Buffer A, pH 8.2

50 mmol Tris-HCl

1 mmol EDTA

Adjust to pH 8.2

Buffer B, pH 8.2

8M Urea

1 mmol Bis-(2-hydroxyethyl)-disulfide

1 mmol 2-Mercaptoethanol

in Buffer A;

Buffer C, pH 8.2

1 mmol Bis-(2-hydroxyethyl)-disulfide

1 mmol 2-Mercaptoethanol

in Buffer A;

Buffer D, ph 8.2

0.6 mol Sodium chloride in Buffer A.

2. Separation and Renaturation of Glu-52-Aprotinin

About 300 mg of freeze dried Lys-15-Glu-52-aprotinin β-galactosidasecyanogenbromide fragments were dissolved in 300 ml buffer B containing300 mg DTT. The solution was reduced for 1 hour at 50° C. under anitrogen atmosphere. Then the solution was applied to a CM-Sephadexcolumn (25×100 mm) filled with about 15 ml CM-sepharose Fast Flow®. Thecolumn was equilibrated with buffer B. The column was washed with bufferB until the baseline was stable. In a first linear gradient elution thecolumn was developed with 100 ml of buffer B and 100 ml of buffer C.Before the second linear elution gradient was applied the column waswashed with buffer A until the baseline was stable. The second gradientwas formed with 100 ml buffer A and 100 ml buffer D. The peak fractionswere tested for trypsin inhibitory activity and by ELISA and WesternBlot (FIG. 10). In a series of experiments the yield estimated by thedifferent tests was in the range of 0.4-2 mg.

3. Purification of Glu-52-Aprotinin by Reversed Phase HPLC

The active fractions were concentrated by evaporation and then dialysedagainst 0.1M NH₄ HCO₃ pH=7.5 for 18 hours. After lyophilization theinhibitor was dissolved in 0.1% TFA and fractionated by reversed phasechromatography on high pore RP-18 column (BIORAD) using a gradient of0.1% TFA in buffer A and 0.1% Trifluoro acetic acid 60% in buffer B. Theinhibitor was characterized by N-terminal sequencing and amino acidanalysis.

EXAMPLE 6 Expression of Val-15-Glu-52-Aprotinin and Characterisation ofthe Product

The fermentation of E. coli transformant with plasmid pRK 49.2.1 and thepurification of the Val-15-Glu-52-aprotinin was performed according tothe same procedures described in Example 5, with the difference that theactivity was tested by an elastase inhibitory assay instead of thetrypsin inhibitory test. The Val-15-Glu-52-aprotinin elutes earlier fromthe CM Sephadex column than the Glu-52-aprotinin.

In a series of experiments the yield estimated by the different testswas 0.1-1 mg.

The same HPLC purification was applied for the isolation ofVal-15-Glu-52-aprotinin. The inhibitory activity was determined byassaying leucocyte elastase inhibition. The inhibitor was characterizedby N-terminal sequencing and amino acid analysis.

EXAMPLE 7 Construction, Expression and Characterization ofArg-15-Glu-52-Aprotinin

For construction of a Arg-15-Glu-52-aprotinin gene, the beta block wasexchanged (FIG. 1), which is the Apa I-Stu I DNA fragment, of theVal-15-Glu-52-aprotinin gene, cloned in plasmid pRK 54.1.1.

This fragment was replaced by a corresponding fragment which codes atamino acid position 15 for Arginine by codon CGT. The resultingrecombinant vector was named pNH 01.1.1, partially sequenced and usedfor further experiments.

At the 5' end of the Arg-15-Glu-52-aprotinin gene a Bam HI site wasadded and the isolated gene was ligated into the Bam HI-Hind III cleavedexpression vector pUR 278.

This DNA was used for transformation of E. coli RR1 delta M15 and atransformant containing the new expression plasmid pRK 112.1.1 wasselected.

From this transformant a β-galactosidase Arg-15-Glu-52-aprotinin fusionprotein was isolated and Arg-15-Glu-52-aprotinin was purified aftercyanogen bromide cleavage as described earlier.

Surprisingly, kinetic studies showed that the recombinantArg-15-Glu-52-aprotinin is a very potent inhibitor of human plasmakallikrein (K_(i) =3.2×10⁻¹⁰ (M)) cationic and anionic human Trypsinwith K_(i) -values below 10⁻¹¹ (M). The K_(i) values were determined bythe method of M. W. Empie and M. Laskowski, Jr., Biochem. 21, 2274(1982).

It will be appreciated that the instant specification and claims are setforth by way of illustration and not limitation, and that variousmodifications and changes may be made without departing from the spiritand scope of the present invention.

What is claimed is:
 1. A pharmaceutical composition containing amicrobially produced aprotinin homolog which is substituted in position52 by an amino acid selected from the group consisting of Glu, Leu, Val,Thr and Ser and containing a pharmaceutically acceptable carrier.
 2. Amethod of treating a patient suffering from an excessive release ofpancreatic elastase, serum elastase or leukocyte elastase comprisingadministering to said patient a therapeutically effective amount of amicrobially produced aprotinin or aprotinin homolog according toclaim
 1. 3. A pharmaceutical composition according to claim 1 containinga microbially produced aprotinin homolog substituted in position 15 by anaturally occuring amino acid.
 4. A pharmaceutical compositioncontaining a microbially produced aprotinin homolog which is substitutedin position 52 by any naturally occurring amino acid except methionineand containing a pharmaceutically acceptable carrier.
 5. Apharmaceutical composition according to claim 3 wherein said amino acidis selected from the group consisting of Arg-15, Val-15, Ile-15, Leu-15,Phe-15, Gly-15, Ser-15, Trp-15, Tyr-15 and Ala-15.
 6. A pharmaceuticalcomposition according to claim 1 wherein the amino acid in position 52is Glu.
 7. A pharmaceutical composition according to claim 1 wherein theamino acid in position 15 is Val or Arg and the amino acid in position52 is Glu.
 8. A pharmaceutical composition according to claim 1 whereinthe amino acid at position 15 is Ile and the amino acid at position 52is Glu.
 9. A pharmaceutical composition according to claim 1 wherein theamino acid at position 15 is Leu and the amino acid at position 52 isGlu.